Time-resolved laser-induced fluorescence spectroscopy systems and uses thereof

ABSTRACT

The invention provides systems for characterizing a biological sample by analyzing emission of fluorescent light from the biological sample upon excitation and methods for using the same. The system includes a laser source, collection fibers, a demultiplexer and an optical delay device. All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of-ordinary skill in the art in which this invention belongs.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/382,791, filed Apr. 12, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/196,354, filed Jun. 29, 2016, now U.S. Pat. No.10,288,567, which is a continuation of U.S. patent application Ser. No.14/776,086, filed Sep. 14, 2015, now U.S. Pat. No. 9,404,870, which is anational stage application of International Application No.PCT/US2014/030610, filed Mar. 17, 2014, which claims the benefit of U.S.Provisional Application No. 61/794,741, filed Mar. 15, 2013; which areincorporated herein by reference for all purposes in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. NS060685awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF INVENTION

The present invention relates generally to techniques for characterizingbiological materials by analyzing laser-induced light emissions fromlabeled or unlabeled biomolecules.

BACKGROUND OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Allen et al., Remington: The Science and Practice of Pharmacy22^(nd) ed., Pharmaceutical Press (Sep. 15, 2012); Hornyak et al.,Introduction to Nanoscience and Nanotechnology, CRC Press (2008);Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology 3^(rd) ed., revised ed., J. Wiley & Sons (New York, N.Y. 2006);Smith, March's Advanced Organic Chemistry Reactions, Mechanisms andStructure 7^(th) ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton,Dictionary of DNA and Genome Technology 3rd ed., Wiley-Blackwell (Nov.28, 2012); and Green and Sambrook, Molecular Cloning: A LaboratoryManual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor,N.Y. 2012), provide one skilled in the art with a general guide to manyof the terms used in the present application. For references on how toprepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2n^(d) ed, Cold Spring Harbor Press (Cold Spring Harbor N.Y., 2013);Kohler and Milstein, Derivation of specific antibody-producing tissueculture and tumor lines by cell fusion, Eur. J. Immunol. 1976 July,6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat. No.5,585,089 (1996 December); and Riechmann et al., Reshaping humanantibodies for therapy, Nature 1988 Mar. 24, 332(6162):323-7.

Laser-induced fluorescence spectroscopy (LIFS) has been extensivelyapplied to complex biological systems to diagnose diseases, such astumors or atherosclerotic plaques, and to analyze chemical orbiochemical composition of organic matters. The benefit of LIFS includesits noninvasive approach to obtain both qualitative and quantitativeinformation of a biological system in vivo. Additional advantages ofLIFS include wavelength tunability, narrow bandwidth excitation,directivity and short pulses excitation. Furthermore, LIFS canselectively and efficiently excite the fluorophores in organic matterand greatly improve the fluorescence selectivity and detectability.

Time-resolved techniques allow real-time evolution of the laser-inducedemission to be directly recorded which was made possible by theavailability of short (nanoseconds) and ultra-short (picoseconds) pulsedlasers, as well as advances in high-speed electronics. Because the lightemission process occurs in a very short time interval after thestimulating event (e.g., fluorescence decay time is in the order ofnanoseconds), the time-resolved measurement can provide informationabout molecular species and protein structures of the sample. Forexample, the time-resolved techniques permit “early” processes(typically the direct excitation of short-lived states or very rapidsubsequent reactions) and “late” processes (typically from long-livedstates, delayed excitation by persisting electron populations or byreactions which follow the original electron process) to be “separated”in the measured data.

The time-resolved measurement only obtains an integrated effect from awide range of wavelengths and can be complemented by spectralinformation in the laser-induced emission to reveal additionalcharacteristics of a sample. To resolve the laser-induced emission intocomponent wavelengths while still being able to perform time-resolvedmeasurement, some existing LIFS techniques use a scanning monochromatorto select wavelengths from the broadband emission one wavelength at atime, and to direct the selected wavelength component to thephotodetector. However, to resolve another wavelength from the emissionspectrum, the sample has to be excited again to produce anotherreemission, while the monochromator is tuned to select the newwavelength.

These existing techniques can take a significant amount of time toresolve multiple spectral components from a wide band light emission.Although each wavelength component can be recorded in real-time, thetransition time of using a monochromator to select another wavelengthcan take up to a few seconds, which becomes the limiting factor inperforming real-time measurements. Furthermore, an overall measurementcan take a large amount of time if a large number of stimulationlocations on the sample have to be measured. Hence, there is a need forsystems and methods that facilitates near real-time recording of bothtime-resolved and wavelength-resolved information from a light emissioncaused by a single excitation of a sample.

SUMMARY OF THE INVENTION

The invention provides a system that characterizes a biological sampleby analyzing light emissions from the biological sample in response toan excitation signal. The system first radiates the biological samplewith a laser impulse to cause the biological sample to produce aresponsive light emission. The system then uses a wavelength-splittingdevice to split the responsive light emission into a set of spectralbands of different central wavelengths. Temporal delays are then appliedto the set of spectral bands so that each spectral band arrives at anoptical detector at a different time, thereby allowing the opticaldetector to temporally resolve the responsive light emission for eachspectral band separately. The delayed spectral bands are then capturedby the system within a single detection window of the optical detector.The captured spectral bands are subsequently processed.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A depicts, in accordance with various embodiments of the presentinvention, a schematic of multi-excitation time-resolved laser-inducedfluorescence spectroscopy. BS: beam splitter; FB: fiber bundle; OD:optical density; LPFW: long pass filter wheel; ExF: excitation fiber;CF: collection fiber; PMT: photo multiplier tube. FIG. 1B shows triggersynchronization.

FIG. 2 depicts, in accordance with various embodiments of the presentinvention, a schematic of an exemplary demultiplexer design.

FIG. 3 depicts, in accordance with various embodiments of the presentinvention, a schematic of the probe.

FIG. 4 depicts, in accordance with various embodiments of the presentinvention, fluorescence emissions of various exemplary biomolecules.

FIG. 5 depicts, in accordance with various embodiments of the presentinvention, a schematic showing the use of continuous NADH monitoring inan ex-vivo brain sample.

FIG. 6 depicts, in accordance with various embodiments of the presentinvention, data demonstrating capabilities of the TRLIFS apparatus tocontinuously monitor the NADH level while the cells are exposed toRotenone, a compound which interferes with NADH-dependent ATPproduction.

In accordance with various embodiments of the present invention, FIG. 7Adepicts the areas observed by the TRLIFS system; FIG. 7B depicts thesample from FIG. 7A is overlapped after treatment with TTC; and FIG. 7Cdepicts the fluorescence intensity plotted for each area (spot).

FIG. 8 depicts, in accordance with various embodiments of the presentinvention, agar/gel with varying concentrations of methotrexate.

In accordance with various embodiments of the present invention, FIG. 9Adepicts fluorescence of MTX at varying concentrations after 20 minsexposure to light with wavelength of 350 nm, and FIGS. 9B-9C depictplots of the fluorescence time course over 20 mins indicating increasein the fluorescence of MTX due to formation of active fluorescent form.

DETAILED DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Allen el al., Remington: The Science and Practice of Pharmacy22^(nd) ed., Pharmaceutical Press (Sep. 15, 2012); Hornyak et al.,Introduction to Nanoscience and Nanotechnology, CRC Press (2008);Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology 3^(rd) ed., revised ed., J. Wiley & Sons (New York, N.Y. 2006);Smith, March's Advanced Organic Chemistry Reactions, Mechanisms andStructure 7^(th) ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton,Dictionary of DNA and Genome Technology 3^(rd) ed., Wiley-Blackwell(Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A LaboratoryManual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor,N.Y. 2012), provide one skilled in the art with a general guide to manyof the terms used in the present application. For references on how toprepare antibodies, see Greenfield, Antibodies A Laboratory Manual2^(nd) ed., Cold Spring Harbor Press (Cold Spring Harbor N.Y., 2013);Kohler and Milstein, Derivation of specific antibody-producing tissueculture and tumor lines by cell fusion, Eur. J. Immunol. 1976 July,6(7):511-9; Queen and Selick, Humanized immunoglohulins, U.S. Pat. No.5,585,089 (1996 December); and Riechmann et al., Reshaping humanantibodies for therapy, Nature 1988 Mar. 24, 332(6162):323-7.

The following description is presented to enable any person skilled inthe art to make and use the invention, and is provided in the context ofa particular application and its requirements. Various modifications tothe disclosed embodiments will be readily apparent to those skilled inthe art, and the general principles defined herein may be applied toother embodiments and applications without departing from the spirit andscope of the present invention. Thus, the present invention is notlimited to the embodiments shown, but is to be accorded the widest scopeconsistent with the claims.

The present invention relates to techniques for characterizingbiological materials by analyzing laser-induced light emissions frombiomolecules (labeled or unlabeled). More specifically, the presentinvention relates to a method and apparatus for characterizingbiological materials by performing a time-resolved andwavelength-resolved analysis on laser-induced fluorescence emissionsfrom the biological materials.

The system described herein may be used for characterizing variousphysiological and disease states, including but not limited to assessingpost-injury tissue viability, tumor and tumor-margin detection,continuous monitoring of cellular metabolism, monitoring blood plasma tooptimize drug therapies. The system can be adapted to variousapplications/uses, depending on the substrate/marker being assayed.

The System

The excitation source is a pulsed laser 100. Output pulses from pulsedlaser radiate upon a biological sample at a predetermined wavelength andpower level that is suitable for exciting biological sample 101 withoutcausing damage to the sample. Pulse laser is controlled by an internalor external pulse controller device or a digital delay device or atrigger device 102, which provides precise timing to each laser impulseoutput. This precise timing is checked at every pulse using a photodiodeand updated using an analog to digital converter device e.g. NIPCIe-6220. In one embodiment, pulsed laser emits ultraviolet (UV) lightpulses to excite biological sample. In another embodiment, pulsed laseremits visible or near infra-red light pulses to excite biologicalsample.

The laser emission from pulsed laser can be coupled/focused into anoptical fiber, and guided to a specific location on biological samplethrough either the optical fiber 103 (FIG. 3) or a lens system.Laser-impulse excitation causes biological sample to produce aresponsive light emission, such as a fluorescence emission, whichtypically has a wide spectrum comprising many wavelengths. Thislaser-induced light emission is then collected by one or morelight-collecting fibers or lenses. In one embodiment of the presentinvention, light-collecting fiber is a bundle of multi-mode fibers 103.In another embodiment the light collecting is achieved using a objectivelens.

Light-collecting fiber then brings the wide band emission light into awavelength-splitting device 104 (FIG. 2), which can comprise one or morewavelength-splitting stages. The wide band emission light undergoes aseries of wavelength splitting processes so that the wide band signalcan be resolved into a number of narrow spectral bands, each with adistinct central wavelength. The wavelength-resolved spectral bands arecoupled into a corresponding delay device 105, which applies apredetermined temporal delay to each spectral band as it travels towardsa photodetector 106. The temporally-delayed spectral bands exiting thedelay device are arranged onto a fast-response photomultiplier tube sothat the fluorescence decay profile of each wavelength-resolved spectralband including the laser light can be individually recorded andtemporally resolved. The delays applied to these spectral bands alloweach optical signal to arrive at the multi-channel plate photomultipliertube (MCP-PMT) at a different time, which allows the decay profile ofeach spectral band detected by the MCP-PMT separately, along with thelaser light. In one embodiment, the output from MCP-PMT can be recordedand displayed using a high-speed digitizer 107. In another embodiment,the output from MCP-PMT can be recorded and displayed using anoscilloscope. In an embodiment, MCP-PMT is a gated, MCP-PMT controlledby a gate control circuit, so that MCP-PMT only responds to lightsignals during a narrow detection window when MCP-PMT is open. In oneembodiment, gate control circuit and pulse control are synchronized sothat all the fluorescence decay profiles associated with a singlelaser-induced excitation may be recorded within a single MCP-PMTdetection window. In an embodiment, the timing of the MCP-PMT gateopening is synchronized to the laser pulse by changing the delay betweenthe laser trigger and MCP-PMT gate using a correction based on theprevious delay. The trigger delay in the laser (the delay between thetrigger signal and actual firing of laser light) is recorded using aphotodiode. The measured trigger delay is used to correct thesynchronization between the laser triggering and MCP-PMT gate. Otherphotodetectors including but not limited to avalanche photodiodes(APDs), silicon PMT, may be used instead of or in addition to MCP-PMTs.The gain of the MCP-PMT can be controlled automatically. In oneembodiment of the present invention the MCP-PMT voltage can bedynamically changed based on the fluorescence signal. In one embodimentof the present invention the voltage change is determined by analyzingthe fluorescence signal and determining the amount of change prior torecording the signal.

The pulsed laser 103 has an inherent delay generating laser light afterthe unit has been externally triggered. In an exemplary embodiment, thedelay in generating the laser light after external delay can be up tobut not limited to 85 microseconds. The delay in triggering the signalhenceforth referred to as ‘trigger delay’ can vary between each pulse oflaser. In order to synchronize the laser light with the PMT gating, theinventors use a photodiode to detect the timing of the laser pulse andcompare it to the external trigger and then correct the timing of nexttrigger based on the last trigger delay (FIG. 1B). In FIG. 1B, t0 iswhen the laser is triggered, t1 is when the laser fires, t2 is when thePMT is triggered and t3 is when the PMT gate turns on. By enabling thisfeedback based trigger synchronization t2 is dynamically set to ensurethat the voltage gain on the MCP-PMT is ‘on’ when the fluorescent signalreaches the MCP-PMT. The digitizer is triggered ‘on’ using a secondphotodiode to ensure smaller data size.

A schematic diagram of the TRLIFS system is depicted in FIG. 1. Invarious embodiments, the system comprises: (i) excitation fibers (ExF),(ii) collection fibers (CF), (iii) a demultiplexer (demuxer), awavelength splitting device that provides micro-measurements about thelifetime of the fluorescence signal, (i.e. the exponential decay of thefluorescence signal), (iv) a photomultiplier tube (MCP-PMT, for example,a high gain (10⁶), low noise and fast rise time detector (−80 ps) suchas Photek 210), (v) an optional preamplifier to provide additional gainafter photomultiplier tube before the signal is digitized, (vi) adigitizer to digitizes the signal received from the Photomultiplier Tube(for example at 6.4 G samples/second), in order to perform data analysis(for example, SP Devices: 108ADQ Tiger), and (vii) a computer system toprocess and display the signal.

The fluorescence signal from the biological tissue can be very high orlow based on the fluorophore in the biological system. The fluorophoreemits fluorescence emission intensity based on the quantum efficiencyand/or absorption of excitation light which may be blocked due tocertain conditions such as the type of sample (for example, tissue,blood, plasma). In order to properly record the fluorescence spectra,the PMT gain needs to be adjusted such that the increased fluorescenceemission does not cause saturation of the signal and low fluorescenceemission does not lead to very low signal to noise ratio. This can beachieved by rapidly changing the voltage across the MCP-PMT based on thepreviously recorded data. In one embodiment, fluorescence light from twopulses of laser is averaged and analyzed (for example, using software)to determine whether the fluorescence signal is too high or too low,after which the voltage across the MCP-PMT (responsible for controllingthe gain of PMT) is changed via communicating between the high voltagepower supply and the computer. In case the fluorescence emission is toohigh the voltage is reduced and vice a versa iteratively till thecorrect amount of signal to noise ratio is achieved. The true signal issaved and analyzed only after the correct SNR is achieved.

In some embodiments, the excitation fiber (for example, a 600 μmdiameter with 0.12 NA, UV grade silica core fiber) connects the lasersource to the sample so as to excite the sample at a desired wavelength.The collection fibers (for example 12 fibers of 200 μm diameter with0.22 NA, UV grade silica core fiber) are packaged into a single bundle;this bundle leads to the demultiplexer (FIG. 3). The 12 fibers can becombined into a single fiber using a technique of combining multi-modefibers in a single fiber. (http://www.ofsoptics.com/). Upon excitationof the sample with a laser at a pre-determined wavelength, thecollection fibers collect the fluorescence signal from the sample, andrelay the signal to the demultiplexer. Various wavelength-splittingfilters in the demultiplexer split the incoming signal based on thewavelengths of the beam splitting devices such as but not limited tofilters or prisms etc. The fluorescence signal pulse (after pulsedexcitation) is relayed to the computer system via the photomultipliertube, the preamplifier and the digitizer, where the fluorescence decayis calculated by deconvolving the (previously recorded) laser pulse fromthe recorded fluorescence pulse.

In various embodiments, the sample is any one or more of blood, plasma,tissue, micriorganisms, parasites, sputum or any other biological samplefrom which the chemical signature can be detected.

Wavelength Splitting Device

FIG. 2 shows schematics of wavelength splitting devices (demultiplexers,demuxers). Laser-induced light emission signal (containing a wide rangeof wavelengths) from the biological sample is collected by alight-collecting fiber, which brings the emitted signal towardswavelength-splitting device.

In an exemplary embodiment of the invention, the biological sample isexcited at wavelengths of about 337-350 nm. In an embodiment, thewavelength splitting device (demultiplexer) depicted in FIG. 1A and FIG.2 splits the incoming signal at wavelengths of: less than 365 nm(excitation wavelength), 365-410 nm, 410-450 nm, 450-480 nm, 500-550 nm,550-600 nm, and greater than 600 nm. As shown in FIG. 1A, the incominglight signal is directed onto the first beam splitting device ofwavelength-splitting device which splits the incoming signal atwavelengths of greater than about 495 nm and less than about 495 nm.After passing through the first beam splitting device, the signal withthe wavelength of greater than 495 nm is focused using a 60 mm focallength biconcave lens and then passes through a second beam splittingdevice that splits the signal at wavelengths of 500-560 nm and greaterthan 560 nm, finally the third beam splitter splits the light in 560-600nm and greater than 600 nm. The signal with wavelength of less than 495nm also pass through a 60 mm focal length biconcave lens a is focusedbefore passing via the fourth beam splitting device that splits the 495nm light signal to wavelengths of about 410-480 nm and less than 410 nm.The light signal with the wavelength of 410-450 nm pass through a fifthbeam splitting device that splits the signal to wavelengths of about415-450 nm and 450-495 nm. The light signals from wavelength less than410 nm goes through a sixth beam splitter and is split in wavelengths365-410 nm and less than 365 nm wavelength, which contains the laserexcitation signal. By recording the laser simultaneously with thefluorescence, it is possible to ensure accurate deconvolution. Thisdemultiplexer design allows detection of biomolecules including but notlimited to flavin mononucleotide (FMN) riboflavin, flavin adeninedinucleotide (FAD) riboflavin, lipopigments, endogenous porphyrin aswell as fluorescence of molecules such as NADH and PLP-GAD in theincoming signals. Beam splitting device mentioned above can be but isnot limited to a dichroic filter, prism, and diffraction grating.

In another exemplary embodiment of the invention, the biological sampleis excited at wavelengths of about 337-350 nm. In this embodiment, thewavelength splitting device splits the incoming signal at wavelengthsof: less than 400 nm, 415-450 nm, 455-480 nm, 400-600 nm and greaterthan 500 nm. Upon exiting light-collection fiber and before enteringwavelength splitting device, the emitted light is first collimated usinga collimating lens. Collimating lens can include, but is not limited to,a Gradient Index (GRIN) lens, or an aspheric lens. The collimated lightbeam is directed onto the first beam splitting device ofwavelength-splitting device which splits the incoming signal atwavelengths of greater than about 400 nm and less than about 400 nm.After passing through the first beam splitting device, the signal withthe wavelength of greater than 400 nm passes through a second beamsplitting device that splits the signal at wavelengths of 400-500 nm andgreater than 500 nm. The signal with wavelength in the range of 400-500nm passes through a third beam splitting device that splits the lightsignal to wavelengths of about greater than 450 nm and less than 450 nm.In various embodiments, signal with wavelengths of less than 450 nm areanalyzed for activities of biomolecules. These wavelengths are importantfor measuring biomolecules including but not limited to free and boundforms of NADH, PLP-GAD or combinations thereof.

By changing the configuration of beam splitting devices spectral bandsof various wavelengths may be detected. Other wavelength bands can beachieved using different sets of filters which will be apparent to theperson of skill in the art.

Temporal Delay Optical Device

As shown in FIG. 1A, each resolved wavelength component from thewavelength-splitting device is coupled to a corresponding delay deviceand subsequently undergoes a predetermined amount of delay in thecorresponding delay device. In various embodiments, the delay devicesare optical fibers with different lengths L1, L2, L3, L4 and so on. In aspecific embodiment, the lengths of the optical fibers may be about 5ft, 55 ft, 115 ft, 165 ft, 215 ft, 265 ft and 315 ft. Other lengths ofoptical fibers may be selected based on the required delay which will beapparent to the person of skill in the art. To temporally separate eachof the wavelength components at the same optical detector, each of thewavelength component travels through a different length of opticalfiber, and thereby experiences a different amount of delay. Eventually,each of the wavelength components arrives at the optical detector atdifferent time which enables each component to be detected separately.

In addition to the length of the optical fiber, other physicalproperties of the optical fiber, including, but is not limited to, therefractive index of the fiber are also used to determine the length ofthe fiber to achieve a specified amount of delay. Since in thetime-domain, each spectral component has a decay profile that lasts fora specific amount of time (e.g., tens of nanoseconds), the temporaldelay between two adjacent spectral components can be designed to besufficiently long to temporally separate the two decay profiles.

In one embodiment of the present invention, the optical detector is agated MCP-MCP-PMT which only responds to incoming light signals within ashort detection window controlled by a gate control circuit. This gatedwindow can be designed to be sufficiently long so that all the resolvedand temporally separated wavelength components will arrive at theMCP-PMT within the gated window. Hence, the gated MCP-PMT can captureall wavelength components which are caused by a single laserinduced-emission within one detecting window. The delay device which isused to temporally separate the resolved spectral bands is not limitedto optical fibers, and any delay device can generally be used.

In various embodiments, the sample is a solid, semi-solid or liquidbiological sample. In various embodiments, the sample is any one or moreof blood, plasma, urine, tissue, microorganisms, parasites, sputum,vomit, cerebrospinal fluid or any other biological sample from which thechemical signature can be detected.

In various embodiments, tissue can be any one or more of prostate, lung,kidney, brain, mucosa, skin, liver, GI tract, colon, bladder, muscle,breast and/or cervix.

The system described herein may be used to detect any molecule that hasa detectable (for example, emitted) signature. In some embodiments, theemitted signature is a fluorescent emission. In some embodiments, thesignature is fluorescence emission decay.

The demultiplexer design described herein allows detection of, forexample, therapeutic agents (labeled or unlabeled), antibodies (labeledor unlabeled), toxin (labeled or unlabeled), endotoxins (labeled orunlabeled), exotoxins (labeled or unlabeled), tumor markers and/or acombination thereof. In various embodiments, unlabeled biomolecules haveintrinsic fluorescence.

The system described herein allows detection of biomolecules includingbut not limited to flavin mononucleotide (FMN) riboflavin, flavinadenine dinucleotide (FAD) riboflavin, lipopigments, endogenousporphyrin as well as fluorescence of molecules such as NADH and PLP-GLDin the incoming signals.

In various embodiments, the therapeutic agents include chemotherapeuticagents. Examples of chemotherapeutic agents include but not limited toAlbumin-bound paclitaxel (nab-paclitaxel), Actinomycin, Alitretinoin,All-trans retinoic acid, Azacitidine, Azathioprine, Bevacizumab,Bexatotene, Bleomycin, Bortezomib, Carboplatin, Capecitabinc, Cetuximab,Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin,Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone,Erlotinib, Etoposide, Fluorouracil, Gefitinib, Gemcitabine, Hydroxyurea,Idarubicin, Imatinib, Ipilimumab, Irinotecan, Mechlorethamine,Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Ocrelizumab,Ofatumumab, Oxaliplatin, Paclitaxel, Panitumab, Pemetrexed, Rituximab,Tafluposide, Teniposide, Tioguanine, Topotecan, Tretinoin, Valrubicin,Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorelbine,Vorinostat, Romidepsin, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP),Cladribine, Clofarabine, Floxuridine, Fludarabine, Pentostatin,Mitomycin, ixabepilone, Estramustine, or a combination thereof. Asdescribed herein, the chemotherapeutic agents may be labeled orunlableled (for example, agents having intrinsic fluorescence). In someembodiments, the label is a fluorescent label. Examples of fluorescentlabels that may be used with the systems, apparatus and methodsdescribed herein to label the therapeutic agents include but are notlimited to indocyanine green (ICG), curcumin, rhodamine (such asrhodamine B, rhodamine 123, rhodamine 6G or variants thereof), greenfluorescent protein (GFP), luciferin, fluorescein, quantum dots or acombination thereof

In various embodiments, antibodies, including therapeutic antibodiesinclude but are not limited to 3F8, 8H9, Abagovomab, Abciximab,Actoxumab, Adalimumab, Adecatumumab, Aducanumab, Afelimomab, Afutuzumab,Alacizumab pegol, ALD518, Alemtuzumab, Alirocumab, Altumomab pentetate,Arnatuximab, Anatumomab mafenatox, Anifrolumab, Anrukinzumab,Apolizumab, Arcitumomab, Aselizumab, Atinumab, Atlizumab, Atorolimumab,Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Belimumab,Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab,Biciromab, Bimagrumab, Bivatuzumab mertansine, Blinatumomab, Blosozumab,Brentuximab vcdotin, Briakinumab, Brodalumab, Canakinumab, Cantuzumabmertansinc, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide,Carlumab, Catumaxomab, cBR96-doxorubicin immunoconjugate, Cedelizumab,Certolizumab pegol, Cetuximab, Citatuzumab bogatox, Cixutumumab,Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Conatumumab,Concizumab, Crenezumab, Dacetuzumab, Daclizumab, Dalotuzumab,Daratumumab, Demcizumab, Denosumab, Detumomab, Dorlimomab aritox,Drozitumab, Duligotumab, Dupilumab, Dusigitumab, Ecromeximab,Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab,Elotuzumab, Elsilimomab, Enavatuzumab, Enlimomab pegol, Enokizumab,Enoticumab, Ensituximab, Epitumomab cituxetan, Epratuzumab, Erlizumab,Ertumaxomab, Etaracizumab, Etrolizumab, Evolocumab, Exbivirumab,Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05, Felvizumab,Fezakinumab, Ficlatuzumab, Figitumumab, Flanvotumab, Fontolizumab,Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab,Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin,Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab,Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tiuxctan, Icrucumab,Igovomab, IMAB362, Imciromab, Imgatuzumab, Inclacumab, Indatuximabravtansine, Infliximab, Inolimomab, Inotuzumab ozogamicin, Intetumumab,Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab,Lambrolizumab, Lampalizumab, Lebrikizumab, Lemalesomab, Lerdelimumab,Lexatumumab, Libivirumab, Ligelizumab, Lintuzumab, Lirilumab,Lodeleizumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab,Mapatumumab, Margetuximab, Maslimomab, Matuzumab, Mavrilimumab,Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab,Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox,Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox,Namatumab, Natalizumab, Nebacumab, Necitumumab, Nerelimomab, Nesvacumab,Nimotuzumab, Nivolumab, Nofetumomab merpentan, Ocaratuzumab,Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab,Onartuzumab, Ontuxizumab, Oportuzumab monatox, Oregovomab, Orticumab,Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab,Pagibaximab, Palivizumab, Panitumumab, Pankomab, Panobacumab,Parsatuzumab, Pascolizumab, Pateclizumab, Patritumab, Pemtumomab,Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin,Pintumomab, Placulumab, Polatuzumab vedotin, Ponezumab, Priliximab,Pritoxaximab, Pritumumab, PRO 140, Quilizumab, Racotumomab, Radretumab,Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab,Reslizumab, Rilotumumab, Rituximab, Robatumumab, Roledumab, Romosozumab,Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab, Sarilumab, Satumomabpendetide, Secukinumab, Scribantumab, Setoxaximab, Sevirumab, SGN-CD19A,SGN-CD33A, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab,Siplizumab, Sirukumab, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab,Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan,Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox, Tefibazumab,Telimomab aritox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab,TGN1412, Ticilimumab (tremelimumab), Tigatuzumab Tildrakizumab, TNX-650,Tocilizumab (atlizumab), Toralizumab, Tositumomab, Tovetumab,Tralokinumab, Trastuzumab, TRBS07, Tregalizumab, Tremelimumab,Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Urelumab, Urtoxazumab,Ustekinumab, Vantictumab, Vapaliximab, Vatelizumab, Vedolizumab,Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Volociximab,Vorsetuzumab mafodotin, Votumumab, Zalutumumab, Zanolimumab, Zatuximab,Ziralimumab, Zolimomab aritox. As described herein, the antibodies maybe labeled or unlabeled. In some embodiments, the label is a fluorescentlabel. Examples of fluorescent labels that may be used with the systems,apparatus and methods described herein to label the therapeutic agentsinclude but are not limited to indocyanine green (ICG), curcumin,rhodamine (such as rhodamine B, rhodamine 123, rhodamine 6G or variantsthereof), green fluorescent protein (GFP), luciferin, fluorescein,quantum dots or a combination thereof.

In various embodiments, toxins include but are not limited to alphatoxin, anthrax toxin, bacterial toxin, diphtheria toxin, exotoxin,pertussis toxin, shiga toxin, shiga-like toxin, heat-stableenterotoxins, channel forming toxins, mycotoxins, cholera toxin,scorpion venom, cholorotoxin and/or tetanus toxins. As described herein,the toxins may be labeled or unlabeled. In some embodiments, the labelis a fluorescent label. Examples of fluorescent labels that may be usedwith the systems, apparatus and methods described herein to label thetherapeutic agents include but are not limited to indocyanine green(ICG), curcumin, rhodamine (such as rhodamine B, rhodamine 123,rhodamine 6G or variants thereof), green fluorescent protein (GFP),luciferin, fluorescein, quantum dots or a combination thereof.

In some embodiments, proteins (for example, cell surface proteins) maybe detected using the system described herein. In some embodiments, theproteins may be detected using antibodies (for example, labeled orunlabeled antibodies) that bind to the cell surface markers. In someembodiments, the proteins may be detected using siRNAs (for example,labeled or unlabeled siRNAs) that bind to the proteins of interest.Examples of proteins that may be detected using the system describedherein include but are not limited to 4-1BB, 5T4, adenocarcinomaantigen, alpha-fetoprotein, anncxin (for example, anncxins A1, A2, A5),BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9(CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (1gEreceptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52,CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP,fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside,glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptorkinase, IGF-1 receptor, IGF-1, IgG1, L1-CAM, IL-13, IL-6, insulin-likegrowth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009,MS4A1, MUCI, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R α,PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1,SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-β,TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 orvimentin. Additional examples include but are not limited to AOC3(VAP-1), CAM-3001, CCLII (cotaxin-1), CD125, CDI47 (basigin), CD154(CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (α chain of IL-2receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, 1gE Fc region, IL-1, IL-12,IL-23, IL-13, IL-17, IL-17A IL-22, IL-4, IL-5, IL-5, IL-6, IL-6receptor, integrin α4, integrin α4β7, Lama glama, LFA-1 (CD11a),MEDI-528, myostatin, OX-40, rhuMAb β7, sclerosein, SOST, TGF beta 1,TNF-α, VEGF-A, beta amyloid, MABT5102A, L-1β, CD3, C5, cardiac myosin,CD41 (integrin alpha-IIβ), fibrin II, beta chain, ITGB2 (CD18),sphingosine-1-phosphate, anthrax toxin, CCR5, CD4, clumping factor A,cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichiacoli proteins, hepatitis B surface antigen, hepatitis B virus, HIV −1,Hsp90, Influenza A hemagglutinin, lipoteichoic acid, Pseudomonasaeruginosa, rabies virus glycoprotein, respiratory syncytial virus,TNF-α, Lewis Y and CEA antigens, Tag72, folate binding protein orcombinations thereof. In some embodiments, the proteins are labeled. Insome embodiments, the label is a fluorescent label. Examples offluorescent labels that may be used with the systems, apparatus andmethods described herein to label the therapeutic agents include but arenot limited to indocyanine green (ICG), curcumin, rhodamine (such asrhodamine B, rhodamine 123, rhodamine 6G or variants thereof), greenfluorescent protein (GFP), luciferin, fluorescein, quantum dots or acombination thereof.

Methods

Based on the combination of the excitation wavelengths and thewavelength-splitting beam splitting devices in the demuxer, fluorescenceof various molecules may be assayed (FIG. 4). For example, withexcitation of the sample at wavelength of 350 nm and appropriatewavelength-splitting beam splitting devices in the demuxer, fluorescenceof biomolecules including but not limited to PLG-GAD(pyridoxal-5′-phosphate (PLP) glutamic acid decarboxylase (GAD)), boundNADH and free NADH, may be assayed. Or, at an excitation of the sampleat wavelength of 440 nm and appropriate wavelength-splitting beamsplitting devices in the demuxer, fluorescence of biomolecules such asFAD (flavin adenine dinucleotide), FMN (flavin mononucleotide) andporphyrins can be assayed.

The invention provides methods for determining tissue viability afterinjury in a subject in need thereof, using the TRLIFS system describedherein. The method includes using the system described herein to measurethe fluorescence emitted from biomolecules (for example, NADH redoxstate) wherein an alteration in the fluorescence signal is indicative oftissue viability. In some embodiments, an alteration in the fluorescencesignal of biomolecules is an increase in the fluorescence signal frombiomolecules in the subject relative to the control (normal) subject. Insome embodiments, an alteration in the fluorescence signal ofbiomolecules is a decrease in the fluorescence signal from biomoleculesin the subject relative to the control (normal) subject. In anembodiment, an alteration in NADH redox state is indicative of tissueviability. In one embodiment, an increase in NADH fluorescence in asubject is indicative of NADH accumulation and poor tissue viability.

The invention also provides methods for monitoring cellular metabolismin a subject in need thereof, using the system described herein. Themethod includes using the TRLIFS system described herein to measure thefluorescence emitted from biomolecules (for example, NADH redox state)wherein an alteration in the fluorescence signal is indicative ofcellular metabolism. In some embodiments, an alteration in thefluorescence signal of biomolecules is an increase in the fluorescencesignal from biomolecules in the subject relative to the control (normal)subject. In some embodiments, an alteration in the fluorescence signalof biomolecules is a decrease in the fluorescence signal frombiomolecules in the subject relative to the control (normal) subject. Inan embodiment, NADH fluorescence may be used to monitor cellularmetabolism. Cellular metabolism may be monitored continuously orperiodically. In various embodiments, continuous monitoring of cellularmetabolism allows, for example, assessment of viability andvulnerability of cells in ischemic condition, effects of drugs (forexample, during drug development or for optimizing therapeutic windows)on cellular metabolism and/or simultaneous monitoring pH and oxygenlevels to determine the metabolic state of the cell.

As described herein, the invention also provides methods for tumordetections using TRLIFS systems described herein.

EXAMPLES Example 1 Continuous Monitoring of Cellular Metabolism

The systems described herein allow continuous monitoring of the changesin the NADH level at very minute scales to determine changes inmetabolic status in response to oxygen depletion, effect ofneuro-protective drugs etc. (FIG. 1A and FIG. 5).

Nicotinamide adenosine dinucleotide (NADH) is involved in redox reactionfor ATP production in aerobic respiration. NADH is produced inmitochondrion during glycolysis and citric acid (TCA) cycle. NADH isoxidized to NAD+ at the mitochondrial membrane producing ATP in theprocess. This process is disrupted in conditions including but notlimited to ischemia due to stroke. In a low oxygen condition, NADHaccumulates in the cell, and persistent oxygen depletion may result incell death, leading to complete breakdown of NADH. These variations inNADH level allow assessment of viability and vulnerability of cells inischemic condition. Fluctuations in NADH levels may be evaluated bymeasuring the fluorescence emission from NADH. NAD+ and NADH both have astrong absorption in UV spectrum, but they differ in their fluorescencecharacteristics. NADH demonstrates strong fluorescence in theviolet/blue band around 440/460 nm of wavelength depending of its bound(to cytochrome) versus free state. Measuring this fluorescence in realtime allows monitoring of changes in the NADH level, assessing themetabolic status of NADH, thereby monitoring cellular metabolism.

In order to excite the tissue, a Q-switched Nd:YaG laser emitting at awavelength of 350 nm was used, running at 1 KHz with a pulse width(FWHM) of 400 ps (Teem Photonics PNVM02510). Total energy per pulse didnot exceed 50 μJ which prevented photo-bleaching of NADH. The excitationlight was delivered to the tissue using a custom made trifurcatedoptical probe. The probe consisted of a central 600 micron fiber fordelivering the excitation light surrounded by twelve 200 micron fibersto collect the fluorescence (FIG. 3). Every other fiber from the twelvecollection fibers were bundled together forming two channels. Onecollection channel/bundle connected to a spectrometer (Ocean Optics,Maya), which measured the fluorescence spectrum every 100 ms and theother channel/bundle connected to a beam splitter (demultiplexer). Thebeam splitter at 452 nm of wavelength separated the significant free andbound fluorescence, which was recorded both by MCP-PMT and spectrometer.

Rabbit brain was removed after sacrificing the animal in the OR andtransported in cold oxygen rich Kreb-ringer solution to the laboratory.The cortex was separated and placed in Kreb-Ringer solution withcontinuous bubbling of the 95% O₂ and 5% CO₂ mixture to keep the tissuealive. The probe was adjusted on the tissue in order to record thefluorescence as shown in FIG. 5. A baseline NADH (bound and free) wasrecorded till the fluorescence from the tissue equilibrated andplateaued. After approximately 30 mins, a measured dose of 50 nMrotenone, which blocks the binding of NADH to cytochrome in themitochondrion, was added. Additional concentrations of rotenone wereadded every 10 mins.

The effect of various concentrations of Rotenone on the rabbit braintissue was recorded (FIG. 6). The results showed that the concentrationsof both free and bound NADH can be mapped in real-time (every ˜100 ms)and response to the external stimuli was recorded. FIG. 6 shows acontinuous plot of NADH fluorescence level over a period of more than 2hours. On adding the 50 nM concentration of Rotenone to the solution, anincrease in NADH level was observed as expected due to blocking on NADHconsumption and subsequent accumulation. As the concentration ofRotenone increased the NADH fluorescence increased as expected. At 80mins time, the gas which was continuously bubbling through the liquidwas stopped and then restarted allowing assessment of the effect ofhypoxia on accumulation of NADH in the tissue and its subsequentconsumption once the oxygen supply was restored. This demonstrated theability of TRLIFS system described herein to monitor the metabolic statein realtime.

Example 2 Determining Tissue Viability After Injury

Recording the NADH levels over a large area of brain after an ischemicstroke permits assessment of the number of viable cells that may be inshock due to lack of oxygen, but may not have undergone apoptosis andthus are salvageable. These cell form the bulk of the region known asthe penumbra and an important goal of stroke treatment is to reduce thesize of penumbra while salvaging as many neurons as possible. MonitoringNADH over the entire penumbra region allows assessment of theeffectiveness of various interventions designed for the same.

In order to excite the tissue, a Q-switched Nd:YaG laser emitting at awavelength 350 nm was used, running at 1 KHz with a pulse width (FWHM)of 400 ps (Teem Photonics PNVM02510). Total energy per pulse did notexceed 50 μJ which prevented photo-bleaching of NADH. The excitationlight was delivered to the tissue using a custom made trifurcatedoptical probe. The probe consisted of a central 600 micron fiber fordelivering the excitation light surrounded by twelve 200 micron fibersto collect the fluorescence. Every other fiber from the twelvecollection fibers were bundled together forming two channels. Onecollection channel/bundle connected to a spectrometer (Ocean Optics,Maya), which measured the fluorescence spectrum every 100 ms and theother channel/bundle connected to a beam splitter (demultiplexer).

A rabbit brain stroke model was used in which a stroke was caused in therabbit brain by injecting a clot in the cerebral artery. The rabbit wassacrificed after testing for neurological damage. The brain was removedand transported to the laboratory in cold O₂ saturated Kreb-RingerSolution. In the laboratory, the infarcted cortex was separated fromrest of the brain and placed in the Kreb-Ringer solution with bubbling95% O₂ and 5% CO₂ mixture. A single reading was recorded from the edgeof the cortex and the probe was moved over the surface of the cortex asshown in FIG. 7A. The fluorescence intensity was recorded from thetissue sample. The tissue sample was submerged in the solution of TTC(2,3,5-triphenyl tetrazolium) which when taken up by the viable cellsturns the cell red. TTC is currently a gold standard for testing theviability of cells. TTC stained tissue was compared to the recordedfluorescence intensity.

A smooth gradient in NADH auto-fluorescence from healthy tissue (redstained area in FIG. 7B) to the dead tissue (unstained area in FIG. 7B)was observed. We also noted that instead of an abrupt change from theviable to dead brain tissue as seen with TTC staining the fluorescenceintensity (FIG. 7C) changed gradually, indicating presence of viablecells in the region indicated as dead.

Example 3 Use of Fluorescence to Determine the Level of Drug/Metabolitein Plasma

Some anticancer drugs are toxic at high dosages and lose their efficacyat lower dosages. This optimal plasma concentration of the drug at whichthe drug is most effective (therapeutic window) varies amongst patientsdue to variation in height, weight, metabolism and ethnicity. In spiteof these variations, currently the drug dosages are calculated based onthe weight of the patient and a standardized pharmacokinetic profile. Aquick and inexpensive method to determine the plasma drug level allowsoptimization of dosages for individual patients. The plasma level ofdrugs may be detected using fluorescence spectroscopy. It is known thatsome of the anticancer drugs such as methotrexate have fluorescentproperties. Herein Applicants show that using the TRLIFS systemsdescribed herein, varying the concentrations of methotrexate (MTX) inagar (FIG. 8) resulted in corresponding change in fluorescence of MTX.

In order to excite the agar gel, a Q-switched Nd:YaG laser emitting atwavelength of 350 nm was used, running at 1 KHz with a pulse width(FWHM) of 400 ps (Teem Photonics PNVM02510). Total energy per pulse didnot exceed 50 μJ which prevents photo-bleaching of NADH. The excitationlight was delivered to the gel using a custom made trifurcated opticalprobe. The probe consisted of a central 600 micron fiber for deliveringthe excitation light surrounded by twelve 200 micron fibers to collectthe fluorescence. Every other fiber from the twelve collection fiberswere bundled together forming two channels. One collectionchannel/bundle goes to a spectrometer (Ocean Optics, Maya), whichmeasure the fluorescence spectrum every 100 ms and the otherchannel/bundle connected to a beam splitter (demultiplexer).

A serial dilution of MTX (25 μg/ml to 25 ng/ml) was prepared in agargel. MTX when exposed to UV light is converted to a more fluorescentform. Upon exposure to UV light the fluorescent form accumulates. Inorder to detect the fluorescent form the conversion from lowfluorescence to fluorescent form was allowed to take place until asaturation level was reached. The final fluorescence intensity wasrecorded and compared to the concentration. The fluorescence intensityof MTX after 20 mins of UV light exposure is a good indicator of theconcentration of MTX in the agar gel as shown in FIGS. 9A-9C.

Example 4 Tumor Detection

Laser-induced fluorescence spectroscopy (LIFS) represents a promisingnew adjunctive technique for in vivo diagnosis. Fluorescencespectroscopy involves exciting the endogenous fluorophores (label-free)within tissues and recording the emission. Fluorescence spectroscopy canby employed in two ways, steady-state or time-resolved fluorescencespectroscopy. The time-resolved measurement resolves fluorescenceintensity decay in terms of lifetimes and thus provides additionalinformation about the underlying dynamics of the fluorescence intensitydecay. Time-resolved measurements are also independent of factors suchas absorption by tissue endogenous fluorophores (e.g blood),photobleaching or any other condition that may affect the fluorescenceintensity. By measuring the fluorescence decay characteristics, whichreflect the differences in the relaxation dynamics of distinctfluorescent molecules, time-resolved measurements have an ability toresolve overlapping spectra, and improve the specificity of thefluorescence measurement.

Applicants show that in patients, the TR-LIFS systems described hereincan discriminate glioma tumors (both high- and low-grade) from thesurrounding normal brain tissue intra-operatively. This work is toestablish the TR-LIFS potential to enhance the ability of theneurosurgeon-neuropathologist team to rapidly distinguish between tumorand normal brain during surgery.

Instrumentation: Experiments were conducted with an instrument setup,which allowed for spectrally-resolved fluorescence lifetimemeasurements. A schematic of the optical and electronic layout of theapparatus is shown in FIG. 1A. Briefly, it consisted of a) A pulsedQ-switch Nd:YaG laser (Teem Photonics, model Teem Photonics PNVM02510,λ=350 nm, pulse width=400 ps FWHM, pulse rate=1 KHz) which was used asthe excitation source, b) a custom made sterilizable trifurcatedfiber-optic probe (Fiberguide, N.J.), c) a gated multi-channel platephoto-multiplier tube (MCP-PMT Photek, UK, model 210, rise time=80 ps)with an optional fast preamplifier (Photek, UK, model PA200-10, 2 GHz),e) a digitizer (ADQ-108, SPDevices, Sveden, 7 Gsamples/sec), and f) acomputer Laptop, g) a custom made demuxer as shown in FIG. 1A andperipheral electronics. The instrument allowed for mobility as it wascontained in a standard endoscopic cart (70×70×150 cm3) internallymodified to accommodate the individual devices. To ensure a very lownoise level from the electronics used such as a high voltage supply andpreamplifier power supply, all the instruments are shielded from themain power supply using a medical grade Isolation transformer (Toroid®ISB-170A).

Delivery catheter: Light delivery and collection were implemented with acustom made bifurcated sterilizable probe. The probe consisted ofnon-solarizing silica/silica step index fibers of 0.11 numericalaperture (NA) (Fiberguide, New Jersey, N.J.). It had a centralexcitation fiber of 600 μm core diameter, surrounded by a collectionring of twelve 200 μm core diameter fibers. All the collection fiberswere bundled together and combined into a single 600 micron fiber. Thecenter-to-center separation between the excitation and collection fiberswas 480 μm. The probe was flexible throughout its entire length (3meters) except of a 7 cm distal part consisted of a rigid stainlesssteel tube. This facilitated the mounting and micromanipulation of theprobe. A spacer with two slits on the opposite sides was added in frontof the distal end of the probe. This allowed the probe to be in contactwith the tissue while maintaining a fixed distance from the tissue. Thetwo slits on the spacer enabled the surgeon to apply a suction tube tomaintain a clear field. The laser light was coupled into theillumination channel of the probe with a standard SMA connector, whilethe distal end of the collection channel was formed into a straight linein order to facilitate coupling to the spectrograph. After tissueexcitation, the emitted fluorescence light was collected and directedinto the entrance slit of the demuxer by bundle one and spectrometer viabundle two. The signal was then detected by the MCP-PMT, amplified bythe fast preamplifier, and finally digitized at 8 bits resolution by thedigital oscilloscope. The overall time resolution of the systems wasapproximately 150 ps.

The fiber optic probe was positioned at 3 mm above the exposed braintissue specimen with the help of a spacer to optimize the probe lightcollection efficiency as previously reported and to steady the probeover the tissue. Time-resolved emission of each sample was recorded atseven separate wavelength bands (355 (<365 nm)), 365-410 nm, 415-450 nm,450-490 nm, 500-560 nm, 560-600 nm and >600 nm) spectral range. Theenergy output of the laser (at the tip of the fiber) for sampleexcitation was adjusted to 5.00/pulse. After the spectroscopic analysisthe tissue was biopsied at the exact site and sent for pathologicalinvestigation.

Each biopsy sample was fixed in 10% buffered formalin. The tissuesamples were fixed on the slides and stained with H&E. All biopsyspecimens were studied by the pathologist and correlated with originalfluorescence spectroscopy measurements results. Histologically, gliomaswere categorized in low grade: Oligodendroglioma,oligodendroastrocytoma, diffuse astrocytoma (WHO Grade II), intermediategrade: anaplastic astrocytoma (WHO Grade III) and high grade: anaplasticoligodendroglioma, anaplastic oligoastrocytoma and glioblastomamultiforme (grade III-IV) based on the WHO grading. For the purpose ofspectroscopic classification in this study the gliomas were grouped aslow grade glioma (LGG) (grade I & II) and high grade glioma (HGG) (gradeIII & IV).

TR-LIFS Data Analysis: In the context of TR-LIFS, the intrinsicfluorescence impulse response functions (IRF), h(n), describes the realdynamics of the fluorescence decay. The IRF were recovered by numericaldeconvolution of the measured input laser pulse from the measuredfluorescence response transients. The Laguerre expansion technique wasused for deconvolution. Laguerre expansion technique was selected overthe more conventional multi-exponential curve fitting for a set ofreasons. It allows for faster deconvolution of the fluorescence IR.Since the Laguerre basis is orthonormal, it provides a unique andcomplete expansion of the decay function. This technique in alsonon-parametric thus does not require a priory assumption of thefunctional form of the decay. Consequently, this allows for theapproximation of fluorescence systems with unknown and complexrelaxation dynamics such as that of biological tissues. This methodallows a direct recovery of the intrinsic properties of a dynamic systemfrom the experimental input-output data. The technique uses theorthonormal Laguerre functions to expand the IRF and to estimate theLaguerre expansion coefficients (LEC). The normalized fluorescencespectra were obtained by dividing the discrete intensities values withthe intensity value at the peak emission. Further, to characterize thetemporal dynamics of the fluorescence decay, two sets of parameters wereused: 1) the average lifetime (τ_(λ)) computed as the interpolated timeat which the IRF decays to of its maximum value; and 2) the normalizedvalue of the corresponding LECs. Thus, a complete description offluorescence from each sample as a function of emission wavelength,λ_(E), was given by the variation of a set of spectroscopic parametersat distinct wavelengths (emission intensity—I_(λ), average lifetime offluorescence emission—τ_(fλ), and Laguerre coefficients LEC_(f)). Thisanalytical approach for characterization of fluorescence decay wasrecently developed by our research group and described in detailelsewhere. Applicants were able to recover the lifetime and Laguerrecoefficient values.

The various methods and techniques described above provide a number ofways to carry out the application. Of course, it is to be understoodthat not necessarily all objectives or advantages described can beachieved in accordance with any particular embodiment described herein.Thus, for example, those skilled in the art will recognize that themethods can be performed in a manner that achieves or optimizes oneadvantage or group of advantages as taught herein without necessarilyachieving other objectives or advantages as taught or suggested herein.A variety of alternatives are mentioned herein. It is to be understoodthat some preferred embodiments specifically include one, another, orseveral features, while others specifically exclude one, another, orseveral features, while still others mitigate a particular feature byinclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be employed invarious combinations by one of ordinary skill in this art to performmethods in accordance with the principles described herein. Among thevarious elements, features, and steps some will be specifically includedand others specifically excluded in diverse embodiments.

Although the application has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the application extend beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses and modifications and equivalents thereof.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe application (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (for example, “such as”) provided withrespect to certain embodiments herein is intended merely to betterilluminate the application and does not pose a limitation on the scopeof the application otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element essential tothe practice of the application.

Preferred embodiments of this application are described herein,including the best mode known to the inventors for carrying out theapplication. Variations on those preferred embodiments will becomeapparent to those of ordinary skill in the art upon reading theforegoing description. It is contemplated that skilled artisans canemploy such variations as appropriate, and the application can bepracticed otherwise than specifically described herein. Accordingly,many embodiments of this application include all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the application unless otherwise indicated herein orotherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications,and other material, such as articles, books, specifications,publications, documents, things, and/or the like, referenced herein arehereby incorporated herein by this reference in their entirety for allpurposes, excepting any prosecution file history associated with same,any of same that is inconsistent with or in conflict with the presentdocument, or any of same that may have a limiting affect as to thebroadest scope of the claims now or later associated with the presentdocument. By way of example, should there be any inconsistency orconflict between the description, definition, and/or the use of a termassociated with any of the incorporated material and that associatedwith the present document, the description, definition, and/or the useof the term in the present document shall prevail.

It is to be understood that the embodiments of the application disclosedherein are illustrative of the principles of the embodiments of theapplication. Other modifications that can be employed can be within thescope of the application. Thus, by way of example, but not oflimitation, alternative configurations of the embodiments of theapplication can be utilized in accordance with the teachings herein.Accordingly, embodiments of the present application are not limited tothat precisely as shown and described.

REFERENCES

Butte, P. V, Fang, Q., Jo, J. A., Yong, W. H., Pikul, B. K., Black, K.L., & Marcu, L. (n.d.). Intraoperative delineation of primary braintumors using time-resolved fluorescence spectroscopy. Journal ofBiomedical Optics, 15(2), 027008. doi:10.1117/1.3374049

Butte, P. V, Pikul, B. K., Hever, A., Yong, W. H., Black, K. L., &Marcu, L. (2005). Diagnosis of meningioma by time-resolved fluorescencespectroscopy. Journal of Biomedical Optics, 10(6), 064026.doi:10.1117/1.2141624

Butte, P. V., Mamelak, A. N., Nuno, M., Bannykh, S. I., Black, K. L., &Marcu, L. (2010). Fluorescence lifetime spectroscopy for guided therapyof brain tumors. NeuroImage, 54, S125 5135.doi:10.1016/j.neuroimage.2010.11.001

Marcu, L., Jo, J. a, Butte, P. V, Yong, W. H., Pikul, B. K., Black, K.L., & Thompson, R. C. (2004). Fluorescence lifetime spectroscopy ofglioblastoma multi forme. Photochemistry and Photobiology, 80, 98-103.doi:10.1562/2003-12-09-RA-023.1

Yong, W. H., Butte, P. V, Pikul, B. K., Jo, J. A., Fang, Q.,Papaioannou, T., . . . Marcu, L. (2006). Distinction of brain tissue,low grade and high grade glioma with time-resolved fluorescencespectroscopy. Frontiers in Bioscience: A Journal and Virtual Library,11(4), 1255-63. Retrieved fromhttp://www.ncbi.nlm.nih.gov/pubmed/16368511

Jo, J. a, Fang, Q., Papaioannou, T., & Marcu, L. (2004). Fast model-freedeconvolution of fluorescence decay for analysis of biological systems.Journal of Biomedical Optics, 9(4), 743 52. doi:10.1117/1.1752919

Lakowicz, J. R. (2006). Principles of fluorescence spectroscopy (3rded., p. xxvi, 954 p.). New York: Springer. Retrieved fromhttp://www.loc.gov/catdir/enhancements/fy0824/2006920796-b.html

Pogue, B. W., Pitts, J. D., Mycek, M. a, Sloboda, R. D., Wilmot, C. M.,Brandsema, J. F., & O'Hara, J. a. (2001). In vivo NADH fluorescencemonitoring as an assay for cellular damage in photodynamic therapy.Photochemistry and Photobiology, 74(6), 817-24. Retrieved fromhttp://www.ncbi.nlm.nih.gov/pubmed/11783938

Schneckenburger, H. (1992). Fluorescence decay kinetics and imaging ofNAD(P)H and flavins as metabolic indicators. Optical Engineering, 31(7),1447. doi:10.1117/12.57704

Sun, Y., Phipps, J., Elson, D. S., Stoy, H., Tinling, S., Meier, J.,Marcu, L. (2009). Fluorescence lifetime imaging microscopy: in vivoapplication to diagnosis of oral carcinoma. Opt Lett, 34(13), 2081-2083.doi:183277 [pii]

The invention claimed is:
 1. A system for characterizing a biologicalsample by analyzing emission of fluorescent light from the biologicalsample upon excitation comprising: (a) a laser source connected to abiological sample via excitation fibers (ExF), wherein the laser isconfigured to irradiate the biological sample with a laser pulse at apredetermined wavelength to cause the biological sample to produce aresponsive fluorescence signal; (b) a light collector, wherein the lightcollector collects the responsive fluorescence signal from thebiological sample, and relays the fluorescence signal to a plurality offilters; and (c) the plurality of filters, each filter of the pluralityof filters being configured to split the responsive fluorescence signalat pre-determined wavelengths to obtain spectral bands, wherein a firstspectral band comprises wavelengths within a range of 410-450 nm, asecond spectral band comprises wavelengths within a range of 450-480 nm,and a third spectral band comprises wavelengths within a range of500-560 nm.
 2. The system of claim 1, wherein a fourth spectral bandcomprises wavelengths within a range of 365-410 nm and wherein a fifthspectral band comprises wavelengths greater than 600 nm, and optionallywherein a sixth spectral band comprises wavelengths of less than 365 nm.3. The system of claim 1, further comprising an optical delay device. 4.The system of claim 3, wherein the optical delay device is adapted tocouple the spectral bands from the plurality of filters into the opticaldelay device, allow the spectral bands to travel through the opticaldelay device, and introduce a controlled time delay to the spectralbands as the spectral bands travel through the optical delay device. 5.The system of claim 3, wherein the optical delay device comprises aplurality of optical fibers and optionally wherein two or more of theplurality of optical fibers have different optical path lengths.
 6. Thesystem of claim 1, wherein the plurality of filters form ademultiplexer.
 7. The system of claim 1, wherein the light collectorcomprises one or more collection fibers (CF) or lenses.
 8. A method forcharacterizing a biological sample by analyzing emission of afluorescence signal from the biological sample upon excitationcomprising: (a) irradiating the biological sample with a laser pulse ata predetermined wavelength to cause the biological sample to produce aresponsive fluorescence signal; (b) collecting the responsivefluorescence signal from the biological sample; and (c) splitting theresponsive fluorescence signal with a plurality of filters, each filterof the plurality of filters being configured to split the responsivefluorescence signal at pre-determined wavelengths to obtain spectralbands, wherein a first spectral band comprises wavelengths within arange of 410-450 nm, a second spectral band comprises wavelengths withina range of 450-480 nm, and a third spectral band comprises wavelengthswithin a range of 500-560 nm.
 9. The method of claim 8, wherein a fourthspectral band comprises wavelengths within a range of 365-410 nm andwherein a fifth spectral band comprises wavelengths greater than 600 nm,and optionally wherein a sixth spectral band comprises wavelengths ofless than 365 nm.
 10. The method of claim 8, wherein the responsivefluorescence signal is emitted by a biomolecule and optionally whereinthe biomolecule is any one or more of PLP-GAD (pyridoxal-5′-phosphate(PLP) glutamic acid decarboxylase (GAD)), bound NADH, free NADH, flavinmononucleotide (FMN) riboflavin, flavin adenine dinucleotide (FAD)riboflavin, lipopigments, endogenous porphyrins or a combinationthereof.
 11. A method for determining tissue viability comprisinganalyzing emission of responsive fluorescence signals from biomoleculesin the tissue by the method of claim 8, wherein an increase inresponsive fluorescence signals of the biomolecule in the biologicalsample relative to a normal sample is indicative of poor tissueviability.
 12. A method for continuously monitoring cellular metabolismcomprising analyzing emission of a responsive fluorescence signal by themethod of claim
 8. 13. A method for determining drug or metabolite levelin plasma comprising analyzing emission of a responsive fluorescencesignal from a biomolecule by the method of claim
 8. 14. The method ofclaim 8, further comprising (d) passing the spectral bands through atime-delay mechanism; (e) obtaining the time-delayed spectral bands; and(f) processing the time-delayed spectral bands.
 15. The method of claim14, wherein processing the time-delayed spectral bands comprisesdetecting the time-delayed spectral bands.